The mechanisms by which exposure to stress triggers mental illness are not understood. Recent work shows that stress activates and modifies PACAP (pituitary adenylate cyclase-activating polypeptide) systems in the rat brain. Mimicking stress-induced increases in PACAP function with a single PACAP treatment causes persistent (lasting more than 1 week) increases in acoustic startle, a measure often used in both preclinical and clinical studies of anxiety. In contrast, a single CRF treatment causes increases in startle that normalize within 24 hr. PACAP's ability to produce long-lasting increases in an anxiety-related behavior in rats differentiates it from CRF and makes it an important new target for stress research. Indeed, new evidence suggests that PACAP is involved in the development of severe and debilitating forms of anxiety in humans, including post-traumatic stress disorder (PTSD), a key sign of which is persistent increases in startle (hyperarousal). This proposal examines the neurobiology of PACAP signaling in stress- and anxiety-related behaviors in rats. Considering the urgent need for new treatments for stress-related disorders, Aim 1 will focus on identifying agents that can block the acute and/or long-lasting behavioral effects of PACAP. We will examine classes of agents that are selective for PACAP (PAC1) receptors and VIP/PACAP (VPAC1) receptors, neither of which has been previously tested in stress studies, as well as kappa-opioid receptor (KOR) antagonists, which have been shown to block stress effects. This work may hasten medication development while providing new directions for mechanistic research. Aim 2 will examine the mechanisms by which PACAP produces persistent effects. Studies focus on the bed nucleus of the stria terminalis (BNST) because (i) the BNST is a major target of PACAP innervation, (ii) stress increases PACAP expression in the BNST, and (iii) infusion of PACAP directly into the BNST produces long-lasting hyperarousal. One set of studies examines how enhancing or disrupting the function of CREB, a downstream target of adenylate cyclase, affects baseline and PACAP-enhanced startle. Another set of studies will extend our new data showing that PACAP but not CRF causes marked downregulation of miR134, a non-coding RNA that negatively regulates neuronal spine density and volume, by examining how enhancing or disrupting miR134 function affects baseline and PACAP-enhanced startle. This work may identify intracellular processes that can be targeted for medication development. Aim 3 will determine if PACAP produces other signs of PTSD, including persistent anhedonia, social withdrawal, deficits in concentration, and impairments in fear extinction. This work may establish that PACAP treatment provides an approach that comprehensively models the myriad symptoms of PTSD. Aim 4 examines sex differences in the strength and persistence of PACAP effects, which may identify still other factors that regulate stress responsiveness. Collectively, the proposed studies may yield insights on the etiology of anxiety disorders and facilitate the development of anti-stress medications.